Sustained antidepressant effect of ketamine through NMDAR trapping in the LHb

Ketamine, an N-methyl-d-aspartate receptor (NMDAR) antagonist1, has revolutionized the treatment of depression because of its potent, rapid and sustained antidepressant effects2–4. Although the elimination half-life of ketamine is only 13 min in mice5, its antidepressant activities can last for at least 24 h6–9. This large discrepancy poses an interesting basic biological question and has strong clinical implications. Here we demonstrate that after a single systemic injection, ketamine continues to suppress burst firing and block NMDARs in the lateral habenula (LHb) for up to 24 h. This long inhibition of NMDARs is not due to endocytosis but depends on the use-dependent trapping of ketamine in NMDARs. The rate of untrapping is regulated by neural activity. Harnessing the dynamic equilibrium of ketamine–NMDAR interactions by activating the LHb and opening local NMDARs at different plasma ketamine concentrations, we were able to either shorten or prolong the antidepressant effects of ketamine in vivo. These results provide new insights into the causal mechanisms of the sustained antidepressant effects of ketamine. The ability to modulate the duration of ketamine action based on the biophysical properties of ketamine–NMDAR interactions opens up new opportunities for the therapeutic use of ketamine.

experiments that demonstrates that Ketamine is actually trapped in the receptors.Structural experiments, single channel experiments and subunit-specific assessment are few of the very important experiments that are missing but necessary to support the conclusions and statements in this work.
Major points 1-The authors employ a model of depression, the CRS, which they have used throughout their previous papers (Yang et al., 2018).The depressive-like phenotype varies across models (stronger anhedonia or behavioural despair depending on the model).Indeed, the authors observe quite of a large variance in the antidepressant efficacy at both 1h and 24h in both FST and SPT.This leads to rather weak statistical effects.Do the authors have any relationship appearing with decay of Ketamine and behaviour?Such a bulk analysis not taking into account individual variability remains outdated compared to the behavioural assertations available with new neurotechnolgies (Cerniauskas et al., 2018).The authors should provide evidence for similar timelines of action in another model of depression, which if possible can have better face validity with the human condition.
Minor point.The authors miss some valuable controls for their behavioural assessment.What happens with treatment non-stress animals?
2-The analysis of the burst is interesting however only expands previous findings.In structures including thalamus or VTA the burst activity relies on the RMP of neurons.The example shown indicate a 10mV difference in RMP.Is it the case that systematically cells bursting are more hyperpolarized?A better biophysical report of parameters should be provided.
When reporting the signatures of burst firing the authors miss to provide the control group.Is the CRS leading to increase bursting.This remains unknown and all data seem to relate only to the ketamine effect on the saline in CRS.Such a control is also missing from the in vivo data.
Minor point.The burst signatures provided in the methods differ from previous work published by the authors, thus this makes difficult to compare across literature.Is there a reason to define it in a different manner?
3-The authors have previously shown that burst are regulated by a complex machine that integrates NMDA receptors, calcium channels and even astrocytes.The authors should show specificity for the effect of Ketamine on NMDA and understand whether the other components are similarly affected.
The measurement of NMDA and AMPA receptors currents and their analysis of I/O curves of absolute value is likely not appropriate.In many instances the currents seem also in the nA range which is at the mercy of space clamp issues.There is no doubt that ketamine can reduce NMDA currents as shown in a variety of studies throughout the brain, however the authors should consolidate these findings in order to eliminate the potential issues of stimulus intensity and location which is difficult to reproduce across recordings.This is particularly the case in a structure that has not an organized anatomical architecture.More experiments, in better controlled conditions should be provided.
Majorly the NMDA component is assessed in this work.Yet, the authors have shown that hypothalamic stimulation strongly affects the AMPA component.Furthermore, the group of R. Malinow has shown the GABA component being affected in a model of depression.The authors need to provide solid evidence for the specificity of their effects.4-The trapping hypothesis proposed by the authors is very intriguing.However, the data fall short to causally demonstrate that ketamine is really trapped within the channel.Furthermore, the trapping idea, as also indicated in their text was previously proposed in other work.This questions the degree of novelty of the study.
What is the rationale in these experiments to use the 100uM dose in Figure 3.The authors nicely show in the initial figures that the real detection of Ketamine was in the order of 16uM.This should be the concentration employed for these experiments as also done in Figure 4. Control experiments monitoring AMPA and GABAa receptors currents are appropriate in this case.An appropriate control showing that Dyngo4a is an efficient blocker of endocytosis of NMDA should be shown as the author cite work using Dynosore rather and in a recombinant system.The authors claim in Figure 4: These results provide a plausible mechanism for the prolonged blockade of NMDARs after ketamine clearance in plasma.Here, the authors fail to explain this statement.The demonstration that endocytosis is not part of the mechanism is poor (controls missing), there is no alternative strategy tested and the trapping hypothesis remains simply a preference.The authors cite a paper that does not support the trapping hypothesis but rather promote the idea that lateral trafficking contribute to the maintenance of NMDA receptors pool.Altogether this renders this work quite preliminary, and descriptive likely appropriate to more specialized audience.

5-
The kick off protocol is intriguing.Also in this case a major control is missing.If the slice is not bathed in ketamine but in saline, and the kick off protocol is provided, is the NMDA component affected.There are instances of plasticity of NMDA in the hippocampus, the VTA and the habenula.This is a major drawback for the claims made by the authors.Is it possible that only postsynaptic activity represents a kick off state?If so, one would think that the conditions to kick off ketamine are larger in a depression state as the activity is higher.This should be tested experimentally.
Why moving to a complete different protocol using higher stimulation in vivo?These presynaptic manipulations can engage plasticity mechanisms at many sites, and this was not controlled for.What the effect of this stimulation with Chrimson at synapses remains completely unknown.
6-The choice and rationale for choosing the hypothalamic input is not clear.The authors published recently the importance of this pathway in stress driven depression.This once again reduces the novelty of the presented work.It would have been instead useful to provide information on the efficiency of multiple inputs onto habenula.The LH is only one among many releasing glutamate.Is the hypothesis valid for every afferent that is excitatory?Minor point.The optogenetic Chrimson-based experiment lacks some controls.What happens if the authors swap the concentrations in vivo, and run the same timed stimulation protocols?It remains unclear why the model works not knowing the stoichiometry of NMDA receptors at every different stage of ketamine presence.Is the Kon-Koff model proposed by the authors better to be tested in heterologous systems enabling the choice of specific subunits.This would provide better information as this basic knowledge is missing in this neuronal circuit.
Minor point -In the introduction the authors state experimental demonstration for the repercussions of habenular bursting on downstream dopamine and serotonin systems.This should be toned down as for the moment it remains a non-tested hypothesis.
-The discussion would need likely some revision.The authors explain their view on how to leverage the trapping mechanisms in the context of therapeutics.This remains far stretched as it is not clear what is the vision of providing negative stimuli to patients.The authors previously indicate the importance of plasticity in the context of stress -now it seems that stress can be leveraged without consequences.This may lead to confusion rather than clarity in the field.

Ma et al.
A. Summary of key results: Ketamine is highly effective for treatment of depression, yet its mechanism of action is not understood: especially the paradox that the behavioral effects of ketamine last about 100 times longer than its half-life for elimination.This study presents the extraordinary finding that the prolonged action of ketamine beyond its tissue concentration lifetime is because of prolonged channel block of the NMDAR receptor by ketamine.B. Originality and significance: To my knowledge the findings are original and highly significant.Now that the the mechanism of ketamine is known, as shown in this study to be prolonged channel block, then it should be possible to devise even more effective prolonged blocking agents.The biophysical observations that support the proposed mechanism of channel block are backed up with appropriate behavioral studies to support the conclusion of prolonged block.This paper should be of wide interest in medicine, neuroscience, and biophysics.C. Data and Methodology: The validity of the approach and the quality of the data support the conclusions drawn.

Referees' comments:
Referee #1: This work follows closely previous publications from the authors.The same group has shown in an outstanding work that ketamine exerts its effect 1h after exposure via affecting burst firing and NMDA receptor function in habenula neurons (Yang et al., 2018; see also Cui et al.,  2019).The authors here increment our knowledge on the understanding how ketamine not only is fast acting but also long-lasting.The study is interesting yet only provides an extension from previous work and a very descriptive sets of results which both reduce the general enthusiasm.Several controls are missing, especially within the electrophysiology experiments.There is a general lack of a solid set of experiments that demonstrates that Ketamine is actually trapped in the receptors.Structural experiments, single channel experiments and subunitspecific assessment are few of the very important experiments that are missing but necessary to support the conclusions and statements in this work.
We appreciate reviewer's positive comment on our previous work.However, we respectfully but strongly disagree with his/her assessment on the novelty of the current manuscript.Below we highlight the novelty of the current findings.
While much progress has been made in understanding ketamine's rapid action, however, much less is understood about mechanisms underlying its sustained effects.Despite a half-life of 13 minutes, ketamine's antidepressant effects in mice can last for at least 24 h.This huge discrepancy poses an interesting basic biological question, and has strong clinical implications.Previously, ketamine's sustained effect was attributed to the de novo spine growth, or the ketamine metabolite HNK.However, ketamine-induced spine growth cannot be detected until 12 hours post treatment, and the half-life of HNK is still less than 30 minutes.In the current work, we reveal a much simpler and more direct mechanism: the trapping blockade of NMDARs.We demonstrate that ketamine continues to block NMDARs in the lateral habenula (LHb), for up to 24 h, long after ketamine's plasma elimination.We further demonstrate that this surprisingly long-lasting molecular inhibition is not due to endocytosis, but governed by the unusually long off-rate of the ketamine-NMDAR interaction.Furthermore, ketamine-suppressed NMDAR currents can be restored in vitro and in vivo upon neural activation and forced NMDAR channel opening.Harnessing these unique biophysical characteristics, we showed that activating the LHb at different ambient ketamine levels ([Kout]) is able to either shorten or extend ketamine's sustained antidepressant effects in vivo.These findings establish a key causal role of the long-term blockade of LHb-NMDARs in mediating ketamine's sustained antidepressant effects, and illustrate a case where a simple molecular biophysical mechanism explains an important therapeutic function.
In addition, our data also resolves a big puzzle in the field as to why other NMDAR inhibitors, such as memantine, have much less optimal antidepressant effects in the clinics.By comparing the trapping property of ketamine with memantine, our study demonstrates that the unique pharmacodynamics property of ketamine is critical in its antidepressant effects, and that optimization of such property is a promising new direction for developing new antidepressant treatment.As pointed out by Reviewer 2, such study should be of broad interest to medicine, neuroscience, and biophysics.
Thanks for the suggestion of structural experiment, single channel experiment, and subunit assessment.However, structural analysis can not reveal trapped ketamine in the channel in the in vivo condition since it requires using proteins purified from in vitro cultured cell lines.Singlechannel recording or subunit assessment can reveal microscopic properties of individual channel or channel composition, but they do not provide more information in terms of drug trapping.Below we provide the control experiments requested, and address your comments in point-bypoint responses.
Major points 1.The authors employ a model of depression, the CRS, which they have used throughout their previous papers (Yang et al., 2018).The depressive-like phenotype varies across models (stronger anhedonia or behavioural despair depending on the model).Indeed, the authors observe quite of a large variance in the antidepressant efficacy at both 1h and 24h in both FST and SPT.This leads to rather weak statistical effects.Do the authors have any relationship appearing with decay of Ketamine and behaviour?Such a bulk analysis not taking into account individual variability remains outdated compared to the behavioural assertations available with new neurotechnolgies (Cerniauskas et al., 2018).The authors should provide evidence for similar timelines of action in another model of depression, which if possible can have better face validity with the human condition.
The reviewer has a good point about tracking the relationship of decay time of ketamine and the behavioral performance at the individual level.However, such experiment would require measuring behavior of the same animal at different time points.Such practice is not recommended for behavioral measure since the tests at the earlier time point will affect the performance at the later time points.That is the exact reason why we chose to use different groups of animals for each time point.The statistics we presented in the paper represents data from several batches of animals and indeed shows significance.Regarding the reviewer's request on a second model of depression, we have now used the WKY rat, which has been demonstrated to be a genetic model of depression 1,2 .As shown in Figure R1 [REDACTED], in the FST assays, the antidepressant effects of ketamine on WKY rats are significant at both the 1 h and 24 h time points.This set of data further confirms ketamine's sustained effects.

Minor point. The authors miss some valuable controls for their behavioural assessment. What happens with treatment non-stress animals?
Thanks for the suggestion.We have now tested the effects of ketamine on naï ve mice.We found that ketamine (i.p. 10 mg kg -1 ) decreased immobile time in the FST at 1 h but not 24 h after injection (Figure R2ac).This can be explained by our use-dependent trapping model of ketamine: the acute swim stress transiently induces LHb burst firing and opens a portion of NMDARs which are blocked by ketamine.However, the number of opened NMDARs by the acute stress is much less than those in the depressive-like state induced by chronic stress.Therefore the effect is no longer significant at 24 h in the naï ve mice.In the SPT test on naï ve mice, there is no acute stress therefore ketamine has no effect even at 1 h (Figure R2d).2.The analysis of the burst is interesting however only expands previous findings.In structures including thalamus or VTA the burst activity relies on the RMP of neurons.The example shown indicate a 10mV difference in RMP.Is it the case that systematically cells bursting are more hyperpolarized?A better biophysical report of parameters should be provided.

[REDACTED]
The reviewer is totally correct that bursting cells in the LHb are more hyperpolarized than non-bursting neurons.This and other detailed analysis of bursting-related biophysical parameters were reported in Fig. 2 and Extended Fig. 6 of our 2018 paper 3 (see figures below).In the current manuscript, we focus on analyzing the LHb burst at different time points, which stands as an important foundation for the mechanistic understanding of ketamine's sustained effects.When reporting the signatures of burst firing the authors miss to provide the control group.Is the CRS leading to increase bursting.This remains unknown and all data seem to relate only to the ketamine effect on the saline in CRS.Such a control is also missing from the in vivo data.
The increase of LHb burst both in vitro and in vivo in the CRS group has been reported in our 2018 paper 3 in Extended Data Fig. 3g-j and Extended Data Fig. 4e-g (see figures below).Consistent with these previous results, in the current manuscript, we also reported that bursting of the CRS group (Fig. 1f) is significantly higher than that of the naï ve control as shown in Extended Data Fig. 2a (P <

Minor point. The burst signatures provided in the methods differ from previous work published by the authors, thus this makes difficult to compare across literature. Is there a reason to define it in a different manner?
We thank the reviewer for the thoughtful comment.In the current manuscript, the onset of a burst remains as two spikes with a maximal inter-spike interval of 20 ms.For the ending of each burst, we changed the cutoff threshold of maximal inter-spike interval from 100 ms in the previous paper to the current 50 ms.Previous papers studying burst firing have used either 100 ms 4-6 or 50 ms 7-9 as the cutoff threshold for burst ending.Reason behind our change is that as we accumulate more in vivo recording data, if we continued to use 100 ms as burst ending threshold, occasionally we got extreme numbers in the "spike number per burst" parameter (>100 spikes per burst, see red arrow in Figure R5j).Therefore we adjusted the criterion to 50 ms.Below we compared major parameters before and after this change (Figure R5).The major conclusions, that ketamine decreased bursting in the LHb in vivo 24 h after injection, is unchanged.3.The authors have previously shown that burst are regulated by a complex machine that integrates NMDA receptors, calcium channels and even astrocytes.The authors should show specificity for the effect of Ketamine on NMDA and understand whether the other components are similarly affected.

Figure R5. Comparison of burst results using old and new criteria. a-d and f-i, Bar graphs illustrating the bursting spike frequency (a, f), bursts per minute (b, g), burst spike percentage (c, h) and spike number per burst (d, i) in CRS mice 24 h after saline or ketamine i.p. injection using current criteria (a-d) and previous criteria (fi). n =
Ketamine's direct molecular target(s) have been previously heavily studied 10 .Among a large number of molecules examined, NMDAR is the one with the highest binding affinity to ketamine 10 .There is no evidence that ketamine can bind the T type calcium channels and astrocytic K channel.That is why we focused our study on NMDAR.As to the specificity issue, as shown in the Figure R6 below, ketamine does not affect the AMPAR or GABAAR components.

The measurement of NMDA and AMPA receptors currents and their analysis of I/O curves of absolute value is likely not appropriate. In many instances the currents seem also in the nA range which is at the mercy of space clamp issues. There is no doubt that ketamine can reduce NMDA currents as shown in a variety of studies throughout the brain, however the authors
should consolidate these findings in order to eliminate the potential issues of stimulus intensity and location which is difficult to reproduce across recordings.This is particularly the case in a structure that has not an organized anatomical architecture.More experiments, in better controlled conditions should be provided.
We totally agree with the reviewer that the absolute value of NMDAR or AMPAR currents alone may be affected by the stimulus intensity and location of the stimulating electrode.That is why we also additionally measured the ratio of NMDA/AMPA (N/A ratio) in Extended Figure 4 (now moved to Fig. 2d, 2j).This ratio normalizes the variability of stimulus intensity and location, and is standardly used in the field to quantify NMDAR or AMPAR responses [11][12][13][14][15] .Importantly, our N/A ratio result also reveals consistent change of NMDAR response by ketamine at the 24 h point.We have readjusted the panels of Regarding the reviewer's concern on space clamp issue, please note that all our recordings of NMDAR-EPSCs are in the pA range, not the nA range.

Majorly the NMDA component is assessed in this work. Yet, the authors have shown that hypothalamic stimulation strongly affects the AMPA component. Furthermore, the group of R. Malinow has shown the GABA component being affected in a model of depression. The authors need to provide solid evidence for the specificity of their effects.
Since ketamine is a well known inhibitor of NMDAR, not of AMPAR or GABAR 10 , we focused our study on NMDA component.Per the reviewer's request, we now also tested ketamine's effects on AMPAR or GABAAR in LHb neurons and confirmed that these two components were not affected (Figure R6).4.The trapping hypothesis proposed by the authors is very intriguing.However, the data fall short to causally demonstrate that ketamine is really trapped within the channel.Furthermore, the trapping idea, as also indicated in their text was previously proposed in other work.This questions the degree of novelty of the study.
We certainly agree that the trapping property of ketamine as a NMDAR blocker has been well documented [16][17][18] and have cited the relevant literatures.However, to our knowledge, nobody has ever proposed that such trapping property can be an important player in ketamine's longterm effect.Thus through this work, we reveal a mechanism hidden in plain sight where a simple molecular biophysical property explains an important therapeutic function of ketamine.Furthermore, building on this, we came up with strategies to either shorten or extend ketamine's sustained antidepressant effects in vivo.As pointed out by Reviewer 2, such study should be of broad interest to medicine, neuroscience, and biophysics.
What is the rationale in these experiments to use the 100uM dose in Figure 3.The authors nicely show in the initial figures that the real detection of Ketamine was in the order of 16uM.This should be the concentration employed for these experiments as also done in Figure 4. Control experiments monitoring AMPA and GABAa receptors currents are appropriate in this case.An appropriate control showing that Dyngo4a is an efficient blocker of endocytosis of NMDA should be shown as the author cite work using Dynosore rather and in a recombinant system.
We thank the reviewer for this comment.Since peak concentration of memantine can reach close to 100 μM in vivo 19 , when comparing the effects of these two drugs in the in vitro experiment in Fig. 3a, we decided to first use the same 100 μM dosage for the two drugs.This result demonstrates that at the same concentration, the two drugs behave differently.Next, exactly to address the reviewer's concern, and to use a dosage close to in vivo ketamine concentration, we lowered the ketamine concentration to 10 μM in Fig. 4, and confirmed that at this in-vivorelevant concentration, ketamine is still trapped for the recorded period.
As demonstrated in Figure R6 above, we now monitored the AMPAR and GABAAR currents during the same recording period, and found them unaltered by ketamine. 20(Figure R7, new Extended Fig. 6).Without Dyngo-4a, LFS successfully induced LTD of NMDAR-eEPSCs (Figure R7a, black dots).In presence of Dyngo-4a, however NMDAR-LTD was strongly suppressed (Figure R7a, red dots), suggesting that Dyngo-4a efficiently blocks endocytosis of NMDAR.We further showed that the NMDAR-eEPSCs can not recover after either 100 μM or 10 μM ketamine washout in the presence of the same concentration of Dyngo-4a (Figure R7b-e).Together these results suggest that the persistent suppression of NMDAR-eEPSCs after ketamine washout is not due to endocytosis.These results have been added to the revised manuscript as Extended Data Fig. 6.The text is also adjusted accordingly (Page 10, Line 215-222).The authors claim in Figure 4: These results provide a plausible mechanism for the prolonged blockade of NMDARs after ketamine clearance in plasma.Here, the authors fail to explain this statement.The demonstration that endocytosis is not part of the mechanism is poor (controls missing), there is no alternative strategy tested and the trapping hypothesis remains simply a preference.The authors cite a paper that does not support the trapping hypothesis but rather promote the idea that lateral trafficking contribute to the maintenance of NMDA receptors pool.Altogether this renders this work quite preliminary, and descriptive likely appropriate to more specialized audience.

To confirm that Dyngo-4a is an efficient blocker of endocytosis of NMDAR, we now tested the effect of Dyngo-4a in a protocol that induces endocytosis of NMDARsthe low-frequency stimulation (LFS)-induced long-term depression (LTD) of NMDAR on brain slices
We thank the reviewer for the comments.Firstly, we have now provided the requested control experiment in the above reply and Figure R7 to rule out the endocytosis mechanism.Secondly, regarding the lateral trafficking, the reviewer is right that the cited paper from the Westbrook group suggests that lateral trafficking could contribute to the recovery of NMDAR-eEPSCs after ketamine inhibition in the hippocampal CA1 neurons.However, in the LHb, there is no such recovery.We have performed a series of experiments comparing this property of LHb and CA1 neurons in a separate manuscript, and now provide the results below.
[REDACTED] 5.The kick off protocol is intriguing.Also in this case a major control is missing.If the slice is not bathed in ketamine but in saline, and the kick off protocol is provided, is the NMDA component affected.There are instances of plasticity of NMDA in the hippocampus, the VTA and the habenula.This is a major drawback for the claims made by the authors.Is it possible that only postsynaptic activity represents a kick off state?If so, one would think that the conditions to kick off ketamine are larger in a depression state as the activity is higher.This should be tested experimentally.
We have now performed the important control experiment that the reviewer suggested, which is to test the kick off protocol on LHb neurons bathed in ACSF without ketamine treatment.We found no change in the NMDAR-eEPSCs (Figure R9  Postsynaptic activity alone is not sufficient for kick-off.Presynaptic glutamate release is also necessary for receptor opening and drug release.Regarding the kick off under the depression state, since the activity is blocked after ketamine treatment, the kick off level would not necessarily be higher than naï ve state.On the other hand, in an experiment we prepare for another manuscript, we found that the level of LHb-NMDAR blockade by ketamine is higher in a depressive-like state than in the naï ve state (Figure R10).

[REDACTED]
Why moving to a complete different protocol using higher stimulation in vivo?These presynaptic manipulations can engage plasticity mechanisms at many sites, and this was not controlled for.What the effect of this stimulation with Chrimson at synapses remains completely unknown.[REDACTED]

As explained in our manuscript
This suggests that a photostimulation-triggered plasticity mechanism is unlikely to contribute to the altered NMDAR-eEPSCs in the ketaminetreated groups.

6.The choice and rationale for choosing the hypothalamic input is not clear. The authors published recently the importance of this pathway in stress driven depression. This once again reduces the novelty of the presented work. It would have been instead useful to provide information on the efficiency of multiple inputs onto habenula. The LH is only one among many releasing glutamate. Is the hypothesis valid for every afferent that is excitatory?
We respectfully disagree that using a published major LHb input as a method to achieve LHb stimulation would reduce the novelty of the current manuscript.The goal here is to have a strong input and activate as many as LHb neurons as possible.As demonstrated in our previous paper, LH is the strongest one among eight LHb input pathways tested.When the LH input was optogenetically stimulated, 100% LHb neurons were activated 21 , making LH an ideal pathway for our manipulation experiment.We would like to emphasize that the experiment here is not to claim the LH as the only endogenous pathway capable of kicking off ketamine, but to use its activity as a stimulation tool to test our hypothesis on ketamine trapping.Therefore we think that testing every afferent pathway is not only beyond the scope of the current manuscript, but would likely defer the purpose by introducing less effective input(s).

Minor point. The optogenetic Chrimson-based experiment lacks some controls. What happens if the authors swap the concentrations in vivo, and run the same timed stimulation protocols?
It remains unclear why the model works not knowing the stoichiometry of NMDA receptors at every different stage of ketamine presence.Is the Kon-Koff model proposed by the authors better to be tested in heterologous systems enabling the choice of specific subunits.This would provide better information as this basic knowledge is missing in this neuronal circuit.
We thank the reviewer for the suggestions.For the experiments in Fig. 5fh, we hoped to test whether ketamine's sustained effects would be shortened by activity kick-off.If we used a lower dosage (5 mg kg -1 ) here, there would not have been a sustained effect at 24 h to start with.Likewise, for the experiments in Fig 5j-l, we hoped to test whether more trapping could extend ketamine's effects.If we had used a high dosage (10 mg kg - 1 ), it would already have caused sustained effect at 24 h.Therefore, we could not swap the concentrations for these two experiments.
The Mameli group has made nice attempts to determine the stoichiometry of NMDA receptors in the LHb by using NR2A and NR2B blocker 22 .There was some complication in the result since the blocked NMDAR currents by both blockers was even smaller than those blocked by one blocker alone.Therefore it was difficult to derive the stoichiometry from the subunit blocker experiment.In addition, we have measured the total NMDAR response at different stage of ketamine presence.This is a more direct and relevant evidence than the stoichiometry information, since our model is about NMDAR blockade.
As discussed in the manuscript, the Kon-Koff dynamics can be very different in the in vitro heterologous system, where the agonist is applied for a much longer duration (second range) than the real presence of glutamate in the in vivo situation (millisecond range).

Minor point In the introduction the authors state experimental demonstration for the repercussions of habenular bursting on downstream dopamine and serotonin systems. This should be toned down as for the moment it remains a non-tested hypothesis.
We thank the reviewer for the suggestion and have now added the word "potentially" in this statement (Page 3, Line 56).

The discussion would need likely some revision. The authors explain their view on how to leverage the trapping mechanisms in the context of therapeutics. This remains far stretched as it is not clear what is the vision of providing negative stimuli to patients. The authors previously indicate the importance of plasticity in the context of stress -now it seems that stress can be leveraged without consequences. This may lead to confusion rather than clarity in the field.
Stress is not always bad, and has been heavily documented to have a Ushape effect on cognition and psychological health 23 .The approach we propose in the discussion utilizes controlled, mild stress within a specific time window.This is different from the chronic, uncontrollable stress that induces depression.

G. References: References have been made to previous work, but because I do not work directly in the fields of depression and ketamine, I would not know if all appropriate references have been made.
H. The paper is very well written and the title, abstract, introduction, results, and discussion are clear and appropriate for the conclusions.

We are very grateful to the reviewer for his/her strong enthusiasm and support! We now address the comments below, and modify the figures and text correspondingly.
No major suggestions.
Minor suggestions are below where the numbers indicate line numbers.

Title: If there is room, spell out LHb
Thank you for the suggestion.We now change the title to "Sustained Antidepressant Effect of Ketamine Through NMDAR Trapping in Lateral Habenula".

Corrected.
99. because the apparent off rate of ketamine is so slow, it seems that 0.23 uM might still contribute to binding.Comment.

Yes. This is indeed the point we would like to make. Because we discovered in this manuscript that the apparent off rate of ketamine is very slow, it can still inhibit the channel at a concentration much lower than its IC50. 0.23 μM is the brain concentration at 1 h after injection. At 24 h after injection, the concentration has become undetectable.
107.There is no e and f in Fig. 1 149.fig. 4  Thank you for the suggestion.We have combined the title and following text into one sentence "Brain concentration of ketamine after a single i.p injection of 5 mg kg -1 ketamine in CRS mice, as measured by LC-MS/MS."

Corrected.
288 An brief explanation of mCherry and ChrimsonR expressing mice would be useful here to interpret the findings.

Laser stimulation (put some brief details here of what laser stimulation was).
We have added the detailed information as: 635 nm, 40 Hz, 2 ms pulse, 250 μW (Page 36, Line 980-981).

Reviewer Reports on the First Revision:
Referees' comments: Referee #1 (Remarks to the Author): In this revised manuscript the authors addressed all the concerns and points raised during the reviewing process.This piece of work adds important cellular information on the effects produced by ketamine and I thank the authors for addressing the points I mentioned both with constructive discussions and new experiments.

Referee #2 (Remarks to the Author):
A-H are unchanged from my previous review.
The authors have suitably addressed my previous review comments.
Additional minor review comments.
Mention "mice" somewhere at the beginning of the Discussion.Perhaps ". . .for up to 24 h in mice (Fig. 1, 2).The data in Figure 1 supports the title in its legend.However Extended Data Figure 1 shows controls, so the data in Extended Data Figure 1 does not support the legend title, which is confusing.Consequently, the title for Extended Data Figure 1 should be followed by a sentence indicating what is actually shown or the title should be changed.
The Extended Data Figure 10 legend may be missing some lines.It needs n values and SEM or SD as appropriate and also indicate the half life in minutes.

Referee #3 (Remarks to the Author):
In this manuscript, the authors aim to reveal the mechanism by which ketamine (but not memantine) exerts its sustained antidepressive effect in people suffering from treatment resistant depression.They use as a model system, mice subjected to 14 days of chronic restraint stress and perform behavioral and electrophysiological tests.Previously, this group showed that antidepressant effect of KET in mice correlates with immediate inhibition of bursting behavior in neurons of the lateral habenula (LHb) neurons (Yang 2018).Here, they investigate the effect of KET at later time points after administration.In Fig 1 they measure the time course for installation and maintenance of antidepressive effects of one KET injection (10 mgs/kg) and they confirm that, as for human patients, depressive-like behaviors (in this case FST, and SPT) in mice decline rapidly (within one hour) and remain suppressed (sustained effect) for at least 24 hours.While this time course mirrors what has been reported for human patients the concentrations used are of concern.Specifically, the half-dose for antidepressant effect observed here is 8 uM (Fig 1a), which corresponds to full anesthesia in humans (Little et al., 1972; Idvall et al.,  1979; Grant et al., 1983; with steady state unconsciousness for levels of 9 uM as reviewed in Zanos 2018).Nevertheless, the authors observe that, as for human patients, although the KET concentration in brain declines rapidly, the antidepressive effect persists at the 24 hr point.This begs the question: if KET was eliminated from plasma within minutes, how can it still exert antidepressive action at 24 hours.This is a long-standing and vexing question in the field and in this manuscript the authors do a good job pointing to the urgency of answering it.They also enumerate the multiple answers that other groups have put forth over the past decade, which NMDA receptor mediated plasticity, and ketamine metabolites (HNK) .Here, the authors concentrate on the firing activity in lateral habenula, a brain area where they have long-standing expertise.Specifically, they test their previous hypothesis that antidepressant effect of KET is mediated by decreased firing of LHB neurons, which is mediated by NMDA receptor inhibition.The authors reported previously that increased bursting activity in this brain region correlates with behavioral depression and the bursting activity can be reduced by NMDA receptor antagonists (ketamine or AP5) but not AMPA receptor antagonists or fluoxetine, a SSRI antidepressant (Yang  2018).In a companion paper (Cui 2018), this group reported that bursting of LHb neurons depends on inputs from (at least) astroglia.Therefore, the activity of the habenula is regulated by extrinsic factors.However, these previous studies did not address a critical point: why only ketamine but not other NMDA receptor inhibitors (such as AP5), which also reduce bursting of the LHb neurons, do not relieve depression?In the present manuscript they compare ketamine and memantine and found that only ketamine produces long-lasting (1 hour) inhibition of NMDA receptors, whereas memantine washes off within 10 minutes.This is an interesting result.Unfortunately, these experiments are done with 100 uM of either KET or MEM, doses that are 100-fold higher than their IC50 for NMDA receptors, and 10-fold larger than the concentration of KET used in anesthesia (10 uM).Nevertheless, the result is interesting and the authors go on to show that following inhibition with 10 uM KET, NMDA receptor activity can be recovered by stimulating neural activity.Which prompts them to propose that the long-lasting effect of KET on inhibiting NMDA receptors is due to 'trapping block'.Here the paper veers away from the previous rigor and into unsubstantiated hypotheses.So far, the existing evidence supports similar mechanisms of action for both memantine and ketamine: open-channel block (with similar IC50~1 uM) (mostly from Jon Johnson's work; Glasgow 2018); and this is supported by structural studies (Zhang 2021 -the Zhu group, Chou 2022 -the Furukawa group).The observation that the ketamine inhibition of NMDA receptor-mediated bursting in LHb can be relieved by stimulation is an observation consistent with persistent openchannel block, but does not come close to a mechanism, or to explaining why KET becomes trapped and MEM does not.One is left to wonder whether this is specific to NMDA receptors endogenous to LHb or to NMDA receptors on neurons that innervate the LHb, or some other mechanism.I must concur with Reviewer 1 that to claim trapping mechanism, one must show experiments with recombinant receptors of distinct composition, single channel recordings from LHb neurons, and some indication that KET remains bound to receptors for long durations (hours!!) is necessary.
In my view, the results presented here represent additional evidence for their previous hypothesis that inhibition of burst firing in LHb correlates with cessation of depressive behaviors.
Additional comments from Referee #2: Reviewer 3's comment is that: "I must concur with Reviewer 1 that to claim trapping mechanism, one must show experiments with recombinant receptors of distinct composition, single channel recordings from LHb neurons, and some indication that KET remains bound to receptors for long durations (hours!!) is necessary.
Reviewer 3 requests evidence at the single channel level for channels of known composition to show long term trapping.The authors did not present such single channel data, but used evoked eEPSCs as an assay to determine the relative percentage of blocked and unblocked NMDARs and also behavioral studies to suggest long term effects after wash out. .The authors less direct approach was consistent with long term trapping and was sufficient for me but not for Reviewer 1 initially and not for Reviewer 3. Note that Reviewer #1 changed their opinion after the authors revised the paper and presented additional data in their response for the revised paper.The question is not whether the authors did the types of experiments the reviewers might like in an isolated model system, but whether the experiments they did in brain and brain slices support their hypothesis of long term trapping, which, in my opinion, they do.Do they prove long term trapping?No. Are scientific questions ever proved?No.The data are either consistent or inconsistent with the hypothesis, and the authors findings support long term trapping in my opinion, but I am not an expert on NMDARs.I am a channel biophysics type.Brief summary of some of the experiments the authors have presented to support prolonged trapping of ketamine (ket) in NMDAR channels.Fig. 1 shows prolonged behavioral effects of ket far beyond the wash out times, assuming the statistics in Fig. 1 k and L are significant which needs to be checked (see statistics below).Prolonged channel block by ket would be consistent with this observation.Fig. 2 shows prolonged effects of ket on reducing the NMDA (but not AMPA) currents in slices.Prolonged channel block of NMDAR by ket would be consistent with this observation.Fig. 3 (100 uM ket or memantine (mem) show that after 10 minute exposure both drugs reduce the NMDAR eEPSCs but that there is recovery from the block over the next hour for mem but not recovery over the next hour for ket, just as would be expected if mem unblocked faster than ket based on previous studies.If the prolonged effects were due to ket and mem being slowly released from reservoirs in the tissue and then reblocking the NMDAR channels, then the results from ket and mem might have been expected to mimic each other, but they did not.Fig. 4 shows that a protocol to open the NMDR channels that would presumably release ket from channel block reduces the inhibition of the NMDAR eEPSCs JUST AS WOULD BE EXPECTED IF KET WERE A VERY LONG LASTING CHANNEL BLOCKER.Fig. 5 shows that an experiment that presumably would untrap ket from NMDA receptors at one hr results in no NMDAR eEPSC reduction at 24 hrs (Fig. 5 d-h), whereas an experiment that would presumably trap additional ket at 10 min leads to reduction in NMDAR eEPSCs at 24 hrs (Fig. 5 i-L), as would be expected if ket were a very long lasting channel blocker.
The authors have presented data to support their hypothesis of long term trapping in real brain and brain slices.Are other explanations possible?Other explanations are always possible in such complex systems, but if one wants to study native receptors in real cells in real brain or brain slices, then the experiments are by necessity more complex in design and interpretation.
Yes, experiments will have to be done in model systems down the road as suggested by Reviewer #3, but these are different types of experiments for a different study.
In light of the concerns of Reviewer #3 I would suggest that the authors be more tempered in their conclusions (and in the title as well) indicating that their findings are consistent with (support) an hypothesis of long term trapping and do not directly establish long term trapping.
Also, in the discussion The authors might also consider the concept of whether delayed diffusion [B.Katz et al.J Physiol.1973 The binding of acetylcholine to receptors and its removal from the synaptic cleft] might or might not play a role.Basically, as KET is released from NMDARs and diffuses out of the synaptic clefts it could likely rebind to unbound NMDARs receptors multiple times greatly extending its effective duration of action.Hence, a binding time of a few hours might be extended to days of action, and observed single channel binding times could be effectively extended many fold in the synaptic cleft through delayed diffusion when not observed in single channel Statistics: In re-reading the paper I found some potential problems with the statistics in a few parts of some figures which I list below that need to be checked by the authors.Of course these concerns could be errors in my measurements and calculations.
A rule of thumb for SEM error bars and unpaired t-tests is that if the SEM error bars overlap or are separated by short distances for the two data sets to be compared, then the differences in means are unlikely to be significant.This can be seen in Fig. 2 where there needs to be gaps between the error bars of the compared data sets to obtain significance, and large gaps to obtain higher significance.
In Fig. 1 K the right most panel for ket blue the undrawn downward error bar would overlap with the upward error bar.I calculate a P ~ 0.25 (not significant) for this while the authors calculate a P of: *** < 0.001, highly significant.(I used a two tailed t-test) This needs to be checked, and of course I may have mis-calculated.
In Fig. 1 K ket middle panel for comparison of baseline and 0-5 min I get a P of about 0.04, not < 0.0001 as the authors get.The other 0 to 1 hr significance levels are also suspect.
In Fig. 1 L middle and right there appear to be similar problems suggesting that the actual P values may be considerably greater than the indicated P values.
At the end of the Fig. 1 legend, the "n = 72, 86 units . . . .and 89, 92 units" needs to be explained or corrected in terms of n and units. .Why are n and units values different?Fig. 4b.For plot to the left of Fig. 4b, state directly if the error bars in this plot come from the 5 and 6 separate cells mentioned for n.State if there is only one 60 minute run for each cell.In Fig. 4b the observed plotted points do not appear to match the stated n's unless there is perfect overlap of some plotted points.If so move overlapping points laterally so they can be seen.Place the SEM bars in Fig. 4b as this is stated as an unpaired t-test.Check the P values in Fig. 4b.
ALL FIGURES NEED SEM BARS if t-tests are applied to the data and all P values need to be checked if the P values do not appear consistent with the SEM bars.
Extended data Figure 3  Thank you.We now added "in mice" in this sentence.
The data in Figure 1 supports the title in its legend.However Extended Data Figure 1 shows controls, so the data in Extended Data Figure 1 does not support the legend title, which is confusing.Consequently, the title for Extended Data Figure 1  We now added the SEM and indicated the half-life to be 13 min in the legend.

Referee #3 (Remarks to the Author):
In this manuscript, the authors aim to reveal the mechanism by which ketamine (but not memantine) exerts its sustained antidepressive effect in people suffering from treatment resistant depression.They use as a model system, mice subjected to 14 days of chronic restraint stress and perform behavioral and electrophysiological tests.Previously, this group showed that antidepressant effect of KET in mice correlates with immediate inhibition of bursting behavior in neurons of the lateral habenula (LHb) neurons (Yang 2018).Here, they investigate the effect of KET at later time points after administration.In Fig 1

SPT) in mice decline rapidly (within one hour) and remain suppressed (sustained effect) for at least 24 hours. While this time course mirrors what has been reported
for human patients the concentrations used are of concern.Specifically, the half-dose for antidepressant effect observed here is 8 uM (Fig 1a), which corresponds to full anesthesia in humans (Little et al., 1972; Idvall et al., 1979; Grant et al., 1983; with steady state unconsciousness for levels of 9 uM as reviewed in Zanos 2018).
We thank the reviewer for bringing up the issue of species differences in drug dosage.It is well known that due to different pharmacokinetics across species, drugs are most often applied at different concentrations in different species 1,2 .For example, for its anesthesia function, ketamine is used at 1-2 mg/kg (corresponding to 60 uM peak concentration) in human 3 , and 100 mg/kg 4 (corresponding to ~ 63 uM peak concentration) in mice 5 .According to the guide for dose conversion between animals and human 2 , using body surface area for dosage conversion, a 10 mg/kg dose in mice would be equivalent to a human dose of ~ 0.8mg/kg, which is close to ketamine's antidepressant dosage for human patients.
In addition, I would also like to point out that in Fig. 1a what we demonstrated is that ketamine's half-life is 13 min, when its concentration has dropped to 8 uM.The concentration only transiently reaches above 8 uM for the initial ~10 min and drops way below this concentration within 1 hr.Our slice experiment in Fig. 3 also tries to mimic this physiological range of drug dynamics by presenting 10 uM ketamine for only 10 min and then wash off the drug.In contrast, for human anesthesia, as shown in the following figure, ketamine concentration is maintained constantly at 10 uM for 2 hrs.Therefore, the antidepressant dose we use here, which is a commonly used antidepressant dose in mice [6][7][8][9] , is much lower than the anesthesia dose in human.
From Idvall, J. et al. 3 Nevertheless, the authors observe that, as for human patients, although the KET concentration in brain declines rapidly, the antidepressive effect persists at the 24 hr point.This begs the question: if KET was eliminated from plasma within minutes, how can it still exert antidepressive action at 24 hours.This is a long-standing and vexing question in the field and in this manuscript the authors do a good job pointing to the urgency of answering it.They also enumerate the multiple answers that other groups have put forth over the past decade, which NMDA receptor mediated plasticity, and ketamine metabolites (HNK) .Here, the authors concentrate on the firing activity in lateral habenula, a brain area where they have long-standing expertise.Specifically, they test their previous hypothesis that antidepressant effect of KET is mediated by decreased firing of LHB neurons, which is mediated by NMDA receptor inhibition.The authors reported previously that increased bursting activity in this brain region correlates with behavioral depression and the bursting activity can be reduced by NMDA receptor antagonists (ketamine or AP5) but not AMPA receptor antagonists or fluoxetine, a SSRI antidepressant (Yang 2018).In a companion paper (Cui 2018), this group reported that bursting of LHb neurons depends on inputs from (at least) astroglia.Therefore, the activity of the habenula is regulated by extrinsic factors.However, these previous studies did not address a critical point: why only ketamine but not other NMDA receptor inhibitors (such as AP5), which also reduce bursting of the LHb neurons, do not relieve depression?In the present manuscript they compare ketamine and memantine and found that only ketamine produces long-lasting (1 hour) inhibition of NMDA receptors, whereas memantine washes off within 10 minutes.This is an interesting result.Unfortunately, these experiments are done with 100 uM of either KET or MEM, doses that are 100-fold higher than their IC50 for NMDA receptors, and 10-fold larger than the concentration of KET used in anesthesia (10 uM).
We thank the reviewer for this comment.As explained in out last round of rebuttal, we first chose 100 μM of KET or MEM for the wash-out experiment since with 10 mg kg -1 i.p. injection, peak concentration of memantine can reach close to 100 μM in vivo 10 .Therefore when comparing the effects of these two drugs in the in vitro experiment in Fig. 3a, we decided to first use the same 100 μM dosage for the two drugs.This result demonstrates that at the same concentration, the two drugs behave differently.Next, exactly to address the reviewer's concern, and to use a dosage close to in vivo ketamine concentration in mice, we lowered the ketamine concentration to 10 μM in Fig. 4, and confirmed that at this in-vivo-relevant concentration, ketamine is still trapped for the recorded period.
Nevertheless, the result is interesting and the authors go on to show that following inhibition with 10 uM KET, NMDA receptor activity can be recovered by stimulating neural activity.Which prompts them to propose that the long-lasting effect of KET on inhibiting NMDA receptors is due to 'trapping block'.Here the paper veers away from the previous rigor and into unsubstantiated hypotheses.So far, the existing evidence supports similar mechanisms of action for both memantine and ketamine: openchannel block (with similar IC50~1 uM) (mostly from Jon Johnson's work; Glasgow 2018); and this is supported by structural studies (Zhang 2021 -the Zhu group, Chou 2022 -the Furukawa group).The observation that the ketamine inhibition of NMDA receptor-mediated bursting in LHb can be relieved by stimulation is an observation consistent with persistent open-channel block, but does not come close to a mechanism, or to explaining why KET becomes trapped and MEM does not.One is left to wonder whether this is specific to NMDA receptors endogenous to LHb or to NMDA receptors on neurons that innervate the LHb, or some other mechanism.
Our data suggests that both drugs are trapped, but ketamine has a much slower off rate.This is consistent with the previous report 11 that despite the same IC50, ketamine has a slower off rate than memantine.

I must concur with Reviewer 1 that to claim trapping mechanism, one must show experiments with recombinant receptors of distinct composition, single channel recordings from LHb neurons, and some indication that KET remains bound to receptors for long durations (hours!!) is necessary. In my view, the results presented here represent additional evidence for their previous hypothesis that inhibition of burst firing in LHb correlates with cessation of depressive behaviors.
Additional comments from Referee #2: Reviewer 3's comment is that: "I must concur with Reviewer 1 that to claim trapping mechanism, one must show experiments with recombinant receptors of distinct composition, single channel recordings from LHb neurons, and some indication that KET remains bound to receptors for long durations (hours!!) is necessary.Reviewer 3 requests evidence at the single channel level for channels of known composition to show long term trapping.The authors did not present such single channel data, but used evoked eEPSCs as an assay to determine the relative percentage of blocked and unblocked NMDARs and also behavioral studies to suggest long term effects after wash out. .The authors less direct approach was consistent with long term trapping and was sufficient for me but not for Reviewer 1 initially and not for Reviewer 3. Note that Reviewer #1 changed their opinion after the authors revised the paper and presented additional data in their response for the revised paper.The question is not whether the authors did the types of experiments the reviewers might like in an isolated model system, but whether the experiments they did in brain and brain slices support their hypothesis of long term trapping, which, in my opinion, they do.Do they prove long term trapping?No. Are scientific questions ever proved?No.The data are either consistent or inconsistent with the hypothesis, and the authors findings support long term trapping in my opinion, but I am not an expert on NMDARs.I am a channel biophysics type.Brief summary of some of the experiments the authors have presented to support prolonged trapping of ketamine (ket) in NMDAR channels.Fig. 1 shows prolonged behavioral effects of ket far beyond the wash out times, assuming the statistics in Fig. 1 k and L are significant which needs to be checked (see statistics below).Prolonged channel block by ket would be consistent with this observation.Fig. 2 shows prolonged effects of ket on reducing the NMDA (but not AMPA) currents in slices.Prolonged channel block of NMDAR by ket would be consistent with this observation.Fig. 3 (100 uM ket or memantine (mem) show that after 10 minute exposure both drugs reduce the NMDAR eEPSCs but that there is recovery from the block over the next hour for mem but not recovery over the next hour for ket, just as would be expected if mem unblocked faster than ket based on previous studies.If the prolonged effects were due to ket and mem being slowly released from reservoirs in the tissue and then reblocking the NMDAR channels, then the results from ket and mem might have been expected to mimic each other, but they did not.

Fig. 4 shows that a protocol to open the NMDR channels that would presumably release ket from channel block reduces the inhibition of the NMDAR eEPSCs JUST AS WOULD BE EXPECTED IF KET WERE A VERY LONG LASTING CHANNEL BLOCKER.
Fig. 5 shows that an experiment that presumably would untrap ket from NMDA receptors at one hr results in no NMDAR eEPSC reduction at 24 hrs (Fig. 5 d-h), whereas an experiment that would presumably trap additional ket at 10 min leads to reduction in NMDAR eEPSCs at 24 hrs (Fig. 5 i-L), as would be expected if ket were a very long lasting channel blocker.
The authors have presented data to support their hypothesis of long term trapping in real brain and brain slices.Are other explanations possible?Other explanations are always possible in such complex systems, but if one wants to study native receptors in real cells in real brain or brain slices, then the experiments are by necessity more complex in design and interpretation.
Yes, experiments will have to be done in model systems down the road as suggested by Reviewer #3, but these are different types of experiments for a different study.
In light of the concerns of Reviewer #3 I would suggest that the authors be more tempered in their conclusions (and in the title as well) indicating that their findings are consistent with (support) an hypothesis of long term trapping and do not directly establish long term trapping.
Again, we are immensely grateful to reviewer 2 for his/her continuous strong support and appreciation of our work!!!
We have now changed the title to "Sustained Antidepressant Effect of Ketamine Potentially via NMDAR Trapping in Lateral Habenula", and changed "establish a causal role" to "support a hypothesis" in discussion (Page15, Line 332).
Also, in the discussion The authors might also consider the concept of whether delayed diffusion [B.Katz et al.J Physiol.1973 The binding of acetylcholine to receptors and its removal from the synaptic cleft] might or might not play a role.Basically, as KET is released from NMDARs and diffuses out of the synaptic clefts it could likely rebind to unbound NMDARs receptors multiple times greatly extending its effective duration of action.Hence, a binding time of a few hours might be extended to days of action, and observed single channel binding times could be effectively extended many fold in the synaptic cleft through delayed diffusion when not observed in single channel Thank you for bringing up this important point, and the insightful suggestion in summary of the Fig. 3 result above.Accordingly, we have now added the following paragraph into discussion: "Another factor that could possibly contribute to ketamine's extended action time in vivo is delayed diffusion 12 .Unlike transmitters which have transporter-based clearance mechanism, ketamine could likely rebind to unbound NMDARs receptors multiple times as it is released and diffuses out of the synaptic clefts or extracellular spaces where astrocytic endfeet tightly wrap around neurons 13 .Given the difference in wash off experiments between memantine and ketamine (Fig. 3), such lateral diffusion seems unlikely to be the major explanation for the sustained effects, but may nevertheless contribute to the extended action time." Statistics: In re-reading the paper I found some potential problems with the statistics in a few parts of some figures which I list below that need to be checked by the authors.Of course these concerns could be errors in my measurements and calculations.
A rule of thumb for SEM error bars and unpaired t-tests is that if the SEM error bars overlap or are separated by short distances for the two data sets to be compared, then the differences in means are unlikely to be significant.This can be seen in Fig. 2 where there needs to be gaps between the error bars of the compared data sets to obtain significance, and large gaps to obtain higher significance.
In Fig. 1 K the right most panel for ket blue the undrawn downward error bar would overlap with the upward error bar.I calculate a P ~ 0.25 (not significant) for this while the authors calculate a P of: *** < 0.001, highly significant.(I used a two tailed t-test) This needs to be checked, and of course I may have mis-calculated.
In Fig. 1 K ket middle panel for comparison of baseline and 0-5 min I get a P of about 0.04, not < 0.0001 as the authors get.The other 0 to 1 hr significance levels are also suspect.
In Fig. 1 L middle and right there appear to be similar problems suggesting that the actual P values may be considerably greater than the indicated P values.
We appreciate the reviewer's careful reading.The data on bursting spike frequency and bursts per min in Fig. 1k,l   At the end of the Fig. 1 legend, the "n = 72, 86 units . . . .and 89, 92 units" needs to be explained or corrected in terms of n and units. .Why are n and units values different?
Thank you for pointing out the need for clarification.As described in Method, "Spiking signals were continuously recorded for 1 h after ketamine treatment (10 mg kg -1 , i.p.) with headstage un-removed.For data collected at 24 h or 3 d after ketamine treatment, since animals need to return to homecage for rest, headstage was removed and remounted".The units cannot be guaranteed to be identical before and after the remounting, and the number of units may also change.Therefore, we used the paired statistical method for the 0-1 h data, and unpaired statistical method for the 24 h and 3 d data.We have added this detailed explanation in Methods (Page39, Line 1111-1116).

ALL FIGURES NEED SEM BARS if t-tests are applied to the data and all P values need to be checked if the P values do not appear consistent with the SEM bars.
We have added SEM bars in Fig. 2d,j,f,l,h,n D. Statistics: Statistics are appropriate.E. Conclusions: The conclusions are well supported by the presented data.F. Improvements: I have no suggested improvements or experiments.G. References: References have been made to previous work, but because I do not work directly in the fields of depression and ketamine, I would not know if all appropriate references have been made.H.The paper is very well written and the title, abstract, introduction, results, and discussion are clear and appropriate for the conclusions.No major suggestions.Minor suggestions are below where the numbers indicate line numbers.1. Title: If there is room, spell out LHb 68. or the ketamine metabolite 99. because the apparent off rate of ketamine is so slow, it seems that 0.23 uM might still contribute to binding.Comment.107.There is no e and f in Fig. 1 149.fig. 4 = Fig. 4 160.fig.= Fig Extended Fig. 8. Rather than repeat the text after the title, perhaps the title should be a conclusion.i.e.Ketamine concentration in the brain falls exponentially with a half time of 12??? minutes or what ever it is.280.folds should be fold 288 An brief explanation of mCherry and ChrimsonR expressing mice would be useful here to interpret the findings.331 suggest: and illustrate a case where a biophysical channel block mechanism extended to extraordinarily long times by the in vivo physiological parameters explains an import therapeutic function.(I suspect you can write a better version.)370 fig.= Fig 924.Laser stimulation (put some brief details here of what laser stimulation was).

Figure R3 .
Figure R3.Bursting-related biophysical parameters (from Fig. 2d, 2e and Extended Fig. 6a-d in Yang et al. 3 ) 0.001, Chi-Square test).These results were described as "The proportion of bursting neurons was significantly higher in CRS mice compared with naï ve mice" (Page 5, Line 111-112 in previous version and Page 5, Line 112-113 in revision).

Figure R4 .
Figure R4.Chronic restraint stress increases burst firing in LHb (from Extended Fig. 3gj and Extended Fig. 4e-g in Yang et al. 3 ) Figure R5.Comparison of burst results using old and new criteria.a-d and f-i, Bar graphs illustrating the bursting spike frequency (a, f), bursts per minute (b, g), burst spike percentage (c, h) and spike number per burst (d, i) in CRS mice 24 h after saline or ketamine i.p. injection using current criteria (a-d) and previous criteria (fi).n = 72, 86 units in 7 mice in saline group and 89, 92 units in 10 mice in ketamine group.e and j, Histogram distribution of spike number per burst of all units in (d) and (i).Pie graph showing percentage of neurons with spike number per burst larger than 30.** P < 0.01; *** P < 0.001; NS, not significant.Error bars indicate SEMs.

Figure R7 .
Figure R7.Recovery of LHb NMDAR-eEPSCs after washout of ketamine in presence of endocytosis blocker Dyngo-4a (30 μM).a, Dyngo-4a successfully blocks LFS (1Hz, 15min)-induced, endocytosis-based longterm depression of NMDAR-eEPSCs in hippocampal CA1 neurons.NMDAR-eEPSCs are isolated by application of PTX and NBQX in Mg 2+ free ACSF under voltage clamp at -70 mV.In the Dyngo-4a group, 30 μM Dyngo-4a is additionally added in ACSF throughout the recording.n = 7 cells for vehicle group n = 9 cells for Dynago-4a group.b, d, In presence of Dyngo-4a, LHb NMDAR-eEPSCs (normalized by baseline) still do not show recovery after wash-out of 100 μM ketamine (b) or 10 μM ketamine (d) in LHb neurons.c, e, Bar graphs showing NMDAR-eEPSCs at the end of the 10 min perfusion period (left) and at 50-60 min (right).100 μM: n = 6 cells for vehicle groups and n = 5 cells for Dynago-4a groups.10 μM: n = 6 cells for vehicle groups and n = 7 cells for Dynago-4a groups.Ket: ketamine.At 10 min: vehicle vs Dyngo-4a, P = 0.70 in (c) and P = 0.09 in (e).At 50-60 min: vehicle vs Dyngo-4a, P = 0.55 in (c) and P = 0.51 in (e).The data of vehicle group in (b) was from the data of ket-group in Figure 3a and the date of vehicle group in (d) was from the data of "no stim" group in Figure 4a.Unpaired t test.NS, not significant.Error bars indicate SEMs.

Figure
Figure R9."Kick off" protocol does not induce NMDAR-LTP.NMDAR-eEPSCs (normalized by baseline) after kick off protocol.NMDAR-eEPSCs are isolated by application of PTX and NBQX in Mg 2+ free ACSF under voltage clamp at -70 mV.n = 7 cells.Error bars indicate SEMs.
(Page12, Line 259-261 in previous version and Page 12 Line 266-268 in revision), to open NMDARs through voltageclamp and zero Mg 2+ condition is only plausible in vitro.Therefore we had to switch to a plausible method that can activate LHb neurons and open their NMDARs in vivo.Activation of a major presynaptic input into the LHb is such a method.For the concern of potential contribution from a plasticity mechanism, our saline-treated control groups did not show difference in either the NMDAR-eEPSCs (Fig. 5g, 5k) or AMPAR-eEPSCs (Figure R11) between the mCherry-and ChrimsonR-expressing groups.
, neuroscience, and biophysics.C. Data and Methodology: The validity of the approach and the quality of the data support the conclusions drawn.D. Statistics: Statistics are appropriate.E. Conclusions: The conclusions are well supported by the presented data.F. Improvements: I have no suggested improvements or experiments.

= Fig. 4 160
. fig. = Fig We have corrected these accordingly.Extended Fig.8.Rather than repeat the text after the title, perhaps the title should be a conclusion.i.e.Ketamine concentration in the brain falls exponentially with a half time of 12??? minutes or what ever it is.
d.The Fig. legend mentions SEM error bars but there are no error bars in this figure.State the test for part d.
should be followed by a sentence indicating what is actually shown or the title should be changed.Thank you for the suggestion.We now changed the legend title to "Single injection of ketamine no longer causes antidepressant effects on day 3 and day 7" (Page29, Line 784-785).The Extended Data Figure10legend may be missing some lines.It needs n values and SEM or SD as appropriate and also indicate the half life in minutes.
are not normally distributed, as verified by D' Agostino-Pearson test, Shapiro-Wik test and Kolmogorov-Smirnov test.Therefore we used nonparametric test (Two way ANOVA Multiple comparisons or Mann-Whitney test), instead of t test here.Compared with the traditional t test method, this nonparametric test focuses more on the distribution characteristics of data (including median and quartiles).As shown in the violin plots below, there is a clear difference between the ketamine group and the saline group.In addition, for the 0-1 h data, we used paired statistical methods.From the scatter plot of Extended Data Fig. 3, it can be seen that ketamine significantly suppressed bursting activity within 1 hour.The P values calculated from these statistical methods are indeed low.Such detailed statistical methods for all the data analysis are now listed in the supplementary table1.

Figure R1 .
Figure R1.Single injection of ketamine causes prolonged suppression of LHb bursting activity in vivo.a, b, Violin plots illustrating the bursting spike frequency (a) and bursts per minute (b) in CRS mice at different time points after saline or ketamine i.p. injection.n = 46 units in 4 mice in saline group and 51 units in 5 mice in ketamine group for 0-1 h data; n = 72, 86

Fig. 4b .
Fig. 4b.For plot to the left of Fig.4b, state directly if the error bars in this plot come from the 5 and 6 separate cells mentioned for n.State if there is only one 60 minute run for each cell.In Fig.4bthe observed plotted points do not appear to match the stated n's unless there is perfect overlap of some plotted points.If so move overlapping points laterally so they can be seen.Place the SEM bars in Fig.4bas this is stated as an unpaired t-test.Check the P values in Fig.4b.
, Fig.3b, Fig.4b, Fig.5g,k,Extended Data Fig.4d-f, Extended Date Fig.6c,e, Extended Data Fig.9e-g and checked all P values.Due to the limitation of legend length, we have now moved all statistical related n values, P values, and methods in legends into the supplementary Table 1.Extended data Figure 3 d.The Fig. legend mentions SEM error bars but there are no error bars in this figure.State the test for part d.

Author Rebuttals to First Revision:
they measure the time course for installation and maintenance of antidepressive effects of one KET injection (10 mgs/kg) and they confirm that, as for human patients, depressive-like behaviors (in this caseFST, and